Use of Quaternary Pyridinium Compounds for Vasoprotection and/or Hepatoprotection

ABSTRACT

The invention relates to a method for the treatment or prevention of diseases or conditions associated with vascular endothelium dysfunction or liver injury comprising the administration to a patient in a need of such treatment or prevention of a therapeutically or prophylactically effective amount of a compound selected from the group consisting of: 
     
       
         
         
             
             
         
       
     
     wherein R represents hydrogen atom, CH 3 , OH, pyridyl (C 5 H 4 N), 1-methylpyridyl (C 5 H 4 N—CH 3 ) or pyridyl substituted with hydroxy group ((OH)C 5 H 3 N), and X represents a physiologically acceptable counterion.

FIELD OF THE INVENTION

The present invention relates to a method of treatment or prevention ofdiseases or conditions associated with endothelium dysfunction or liverinjury. In particular, the method comprises the administration ofcertain quaternary pyridinium compounds to a subject in a need of suchtreatment.

The invention is based on the unique ability of certain quaternarypyridinium compounds to stimulate endogenous production and release ofprostacycline PGI₂ in vascular endothelium which can bring therapeuticeffects in diseases associated with endothelial dysfunction, oxidativestress and insufficient endothelial PGI₂ production, as well as in theconditions of enhanced risk of incidence of said diseases(vasoprotective effect). The use of such compounds can provide healingand preventive therapeutic effects among others in diabetes, metabolicsyndrome, athero-sclerosis and also in liver injuries (hepatoprotectiveeffect).

BACKGROUND ART

There is increasing evidence that endothelial dysfunction plays a keyrole in the formation and progression of atherosclerotic plaque as wellas in the development of diabetes and diabetic complications.Endothelial dysfunction has recently gained diagnostic, prognostic andtherapeutic significance in atherothrombosis and diabetes (Heitzer T,Schlinzig T, Krohn K, Meinertz T, Munzel T. Endothelial dysfunction,oxidative stress, and risk of cardiovascular events in patients withcoronary artery disease. Circulation 2001;104:2673-2678; Schachinger V,Britten M B, Zeiher A M. Prognostic impact of coronary vasodilatordysfunction on adverse long-term outcome of coronary heart disease.Circulation 2000;101:1899-1906; Perticone F, Ceravolo R, Pujia A,Ventura G, Iacopino S, Scozzafava A, Ferraro A, Chello M, MastrorobertoP, Verdecchia P, Schillaci G. Prognostic significance of endothelialdysfunction in hypertensive patients. Circulation 2001; 104:191-196;Suwaidi J A, Hamasaki S, Higano S T, Nishimura R A, Holmes D R, Jr.,Lerman A. Long-term follow-up of patients with mild coronary arterydisease and endothelial dysfunction. Circulation 2000;101:948-954), deJager J, Dekker J M, Kooy A, Kostense P J, Nijpels G, Heine R J, BouterL M et al.: Endothelial dysfunction and low-grade in-flammation explainmuch of the excess cardiovascular mortality in individuals with type 2diabetes: the Hoorn Study. Arterioscler Thromb Vasc Biol, 2006, 26,1086-1093).

Endothelial dysfunction is defined as a functional endothelium conditionwhich is characterized by deficiency of vasoprotective factors andenhanced production of pro-coagulative and pro-inflammatory factors(Chlopicki S, Kardiologia po Dyplomie, 2005, Vol. 4 No. 5, 77-88).Clinically, endothelial dysfunction is identified as the impairement ofbiological activity of NO, which is diagnosed as the impairment ofvasodilating NO activity. Monitoring of bioavailability of NO ispossible by measurement of NO-dependent vasoconstrictive endotheliumfunction in vivo (Chlopicki S, Kardiologia po Dyplomie, 2005, Vol. 4 No.5, 75-81). The impairment of NO activity coincides with oxidant stress(Heitzer T, Schlinzig T, Krohn K, Meinertz T, Munzel T. Endothelialdysfunction, oxidative stress, and risk of cardiovascular events inpatients with coronary artery disease. Circulation 2001;104:2673-2678)and impairment of PGI₂ synthesis (Kyrle P A, Minar E, Brenner B, EichlerH G, Heistinger M, Marosi L, Lechner K. Thromboxane A₂ and prostacyclingeneration in the microvasculature of patients withatherosclerosis—effect of low-dose aspirin. Thromb Haemost1989;61:374-377), although systemic level of PGI₂ may be elevated.Indeed, it has been proposed that increased lipid peroxidation mightpromote development of atherosclerosis owing to selective impairment ofprostacyclin synthesis in endothelial cells and subsequent activation ofplatelets (Gryglewski R J. Prostacyclin and atherosclerosis. TIPS 1980;1: 164-168; Gryglewski R J. Prostaglandins, platelets, andatherosclerosis. CRC Crit Rev Biochem 1980;7:291-338; Gryglewski R J,Szczeklik A. Prostacyclin and atherosclerosis—experimental and clinicalapproach. 1983;213-226). This concept was then supported experimentally.It is apparent now that impairment of PGI₂ synthesis in endothelium maylead to the excessive stimulation of TP receptors in endothelium andvascular smooth muscle cells by TXA₂, PGH₂ or other eicosanoids. Suchmechanisms lead to subsequent vasoconstriction, platelet activation,inflammatory response of endothelium as well as endothelial apoptosis(Chlopicki S, Gryglewski R J. Endothelial secretory function andatherothrombosis in “The Eicosanoids”, chapter 23, 267-276. ed. P.Curtis-Prior, John Wiley and Sons, Ltd, 2004). This means that theimpairment of PGI₂ synthesis in endothelium may trigger or enhanceinflammatory and thrombotic processes in vascular wall, which are nowconsidered to be the key elements of atherosclerosis. Endothelialdysfunction plays also the key role in the development of diabetes (deJager J, Dekker J M, Kooy A, Kostense P J, Nijpels G, Heine R J, BouterL M et al.: Endothelial dysfunction and low-grade inflammation explainmuch of the excess cardiovascular mortality in individuals with type 2diabetes: the Hoorn Study. Arterioscler Thromb Vasc Biol, 2006, 26,1086-1093.) Therefore, the result of the ability of certain quaternarypyridinium salts to stimulate endothelial PGI₂ synthesis can be theirpotential anti-atherosclerotic and anti-diabetic effects. Similarly, inmany other diseases wherein endothelial dysfunction plays a role inpathogenesis, pharmacological enhancement of PGI₂ synthesis inendothelium caused by said compounds may bring therapeutic effects. Someof quaternary pyridinium salts can also have therapeutic effects indiseases responding to prostacycline analogs, such as for example liverinjuries, pulmonary hypertension, and wound healing.

The use of quaternary pyridinium salts of the formula:

wherein R is NH₂, CH₃, or N(H)CH₂OH group, and X is pharmaceuticallyacceptable counterion, for the preparation of vasoprotective agent forthe treatment or prevention of conditions or diseases associated withdysfunction of vascular endothelium, oxidative stress, and/orinsufficient production of endothelial prostacyclin PGI₂ is described inWO2005/067927. In particular, anti-atherosclerotic and thrombolyticactivity of the compounds of the above formula was shown.

In publications of R. Stadler et al. in. w J. Agric. Food Chem. 2002,50, 1192-1199 and 1200-1206 products of trigonelline decompositionformed during coffee roasting were reported. Reportedly, the mainnon-volatile products of trigonelline pyrolysis were 1-methylpyridineand dialkylpyridinium compounds. Potential anticancer activity ofalkylpyridinium compounds, especially 1-methylpyridine, was suggested.

SUMMARY OF THE INVENTION

In brief, the invention relates to a method for the treatment orprevention of diseases or conditions associated with vascularendothelium dysfunction or liver injury comprising the administration toa patient in a need of such treatment or prevention of a therapeuticallyor prophylactically effective amount of a compound selected from thegroup consisting of:

wherein R represents hydrogen atom, CH₃, OH, pyridyl (C₅H₄N),1-methylpyridyl (C₅H₄N—CH₃) or pyridyl substituted with hydroxy group((OH)C₅H₃N), and X represents a physiologically acceptable counterion.

BRIEF DESCRIPTION OF THE FIGURES OF THE DRAWING

FIG. 1 shows the scheme of the method for detection of thrombolyticaction of drugs in vivo in rats.

FIG. 2 illustrates the lack of significant thrombolytic response afterintravenous administration of nicotinamide or nicotinic acid in vivo (30mg/kg).

FIG. 3 illustrates the lack of significant thrombolytic response afterintravenous administration of trigonelline in vivo (30 mg/kg).

FIG. 4 shows the thrombolytic response induced by intravenousadministration of trigonelline thermolysate in vivo (30 mg/kg).

FIG. 5 shows the thrombolytic response in vivo induced by intravenousadministration of 1-methylpyridinium chloride (30 mg/kg) (upper record)and 1,4-dimethylpyridinium chloride (30 mg/kg) (lower record).

FIG. 6 shows the effect of trigonelline thermolysate on postprandialhyperglycemia in comparison with metformine and non-treated control.

FIG. 7 shows the hepatoprotective effect of 1,4-dimethylpyridiniumchloride in vivo.

DETAILED DISCLOSURE OF THE INVENTION

The aim of the present invention was to provide new therapeutic agent,having vasoprotective and/or hepatoprotective activity and therefore thepotential utility for the treatment and prevention of among othersatherosclerosis, thrombosis, diabetes, metabolic syndrome, and liverinjuries of different origin.

It has been unexpectedly and surprisingly found that some compounds thatare formed during the process of thermal decomposition of trigonellineshow vasoprotective activity as well as hepatoprotective activity due totheir ability to improve endothelial function by stimulation of therelease of endogenous prostacycline.

Accordingly, a subject of the invention is a method for the treatment orprevention of diseases or conditions associated with vascularendothelium dysfunction or liver injury comprising the administration toa patient in a need of such treatment or prevention of a therapeuticallyor prophylactically effective amount of a compound selected from thegroup consisting of:

wherein R represents hydrogen atom, CH₃, OH, pyridyl (C₅H₄N),1-methylpyridyl (C₅H₄N—CH₃) or pyridyl substituted with hydroxy group((OH)C₅H₃N), and X⁻ represents a physiologically acceptable counterion.

As defined above, X⁻ may be any organic or inorganic physiologicallyacceptable anion, i.e. such an anion which is devoid of any toxic orotherwise harmful effect to a body, in particular acceptable for oraladministration in therapeutic doses.

Non-limiting examples of suitable physiologically acceptable inorganicanions are chloride, bromide, iodide and carbonate. Non-limitingexamples of suitable physiologically acceptable organic anions areanions of mono-, di- and tricarboxylic acids, for example acetate,benzoate, salicylate, hydroxyacetate, lactate, malonate and citrate,etc.

Preferred physiologically acceptable anion is chloride.

In one of the embodiments the compound has the formula

wherein R represents hydrogen atom or CH₃ in the position 2 or 4 of thepyridine ring.

One of the preferred embodiments is the compound of the formula

wherein R represents CH₃ in the position 4 of the pyridine ring.

Particular compounds useful in the method of the invention are1,4-dimethylpyridine salts, especially chloride, lactate or citrate,especially advantageously 1,4-dimethylpyridine chloride of the formula:

Another particular compounds useful in the method of the invention are1,2-dimethylpyridine salts, especially chloride, lactate or citrate,especially advantageously 1,2-dimethylpyridine chloride of the formula:

Yet another particular compounds useful in the method of the inventionare 1-methylpyridine salts, especially chloride, lactate or citrate,especially advantageously 1-methylpyridine chloride of the formula:

Pyridinium compounds of formulae (I), (II) and (III) can be obtained bychemical synthesis, using conventional techniques known for a personskilled in the art of organic synthesis.

Said compounds can be for example obtained starting from correspondingknown compounds non-substituted with methyl group at the nitrogen atomof the pyridine ring, i.e. at position 1 of the pyridine ring. When X⁻represents halogenide anion, compounds of formulae (I), (II) and (III)can be obtained by direct methylation with methyl halogenide fromstarting materials non-substituted with methyl group at the nitrogenatom, in a manner known per se. Compounds wherein X⁻ represents chlorideanion can be for example obtained by treating with methyl chloride astarting compound non-substituted with methyl group in position 1, asdescribed for example in AT 131,118, GB3 48345, U.S. Pat. No. 3,614,408,and U.S. Pat. No. 4,115,390.

Compounds wherein X⁻ is an anion other than halogenide can be obtainedby the exchange of halogenide anion with another anion, for example bythe treatment with a salt of such another anion, for example silversalt. For example, lactates or acetates of the above compounds can beobtained by treating a halogenide, preferably chloride, with a salt ofanother anion, for example silver salt. For example, lactates oracetates of the above compounds can be obtained by treating ahalogenide, preferably chloride, with solver lactate or acetate,respectively.

The above compounds of formulae (I), (II) and (III) are produced in aprocess of thermal decomposition (thermolysis) of trigonelline(N-methyl-nicotinic acid, 3-carboxy-1-methylpyridinium salt). Individualcompounds of formulae (I), (II) and (III) can be therefore obtained alsoby separation of the products of pyrolysis of trigonelline(thermolysate), obtained for example by pyrolysis in the oxygen-freeatmosphere at about 220° C.

Thus, in one of embodiments of the method of the invention a mixture ofthe compounds selected from the above defined compounds of formulae (I),(II) and (III) can be used, as comprised in the product of thermaldecomposition of trygonelline, i.e. trigonelline thermolysate.

It has been found that pyridinium compounds defined above posess uniquepharmacological properties due to their ability to release endogenousprostacycline (PGI₂) from endothelium. In consequence, the compounds canimprove tissue perfusion, exert anti-thrombotic, thrombolytic,anti-apoptotic or antisclerotic activity, protect gastrointestinalmucosa, as well as exert hepatoprotective activity in the treatment orprevention of liver injuries.

The advantage of the above pyridinium compounds is the fact that theirvasoprotective activity is most probably not accompanied by anyhypotensive action. Furthermore, thrombolytic activity is not connectedwith direct action on blood platelets. The above pyridinium compounds donot have any direct action on leucocytes activity.

Without any wish to be bound by theoretical considerations, the presentinventors believe that the compounds defined in the present invention,being endowed with a positive charge, bound through electrostaticinteractions with anionic compounds present on the surface of vascularendothelium, such as glycosaminoglycans. The consequences of suchbonding can be several endothelial effects, which effects can havepositive influence from the pharmacological point of view. Among sucheffects can be the release of NO and/or prostacycline, due to whichendothelial dysfunction can be improved and conditions or diseasesassociated with such dysfunction can be treated or prevented.

In one of the aspects of the method of the invention such conditions ordiseases associated with endothelial dysfunction are those associatedwith endothelial dysfunction accompanied by hypercholesteremia,hypertriglycerydemia or low HDL cholesterol level.

In one of particularly preferred embodiments of the above aspect diseaseor condition is thrombosis or atherosclerosis.

In another aspect of the method of the invention said disease orcondition can be an acute cardiovascular event associated withatherosclerosis, in particular sudden cardiac death, acute coronarysyndrome (including unstable coronary artery disease, myocardialinfarct), a condition which necessitates the coronary angioplasty (PCI)or coronary-aortal by-pass surgery (CABG), ischemic stroke, surgicaltreatment with extracorporeal circulation or a condition whichnecessitates a peripheral circulation revascularization.

In another aspect of the method of the invention said condition ordisease is atherosclerosis in a patient with stable coronary disease,brain ischemic episodes or atherosclerosis of extremities, includingBurger disease.

In yet another aspect of the method of the invention said condition ordisease is selected from the risk factors of atherosclerosis selectedfrom the group consisting of: hypercholesterolemia, arterialhypertension, smoking, hyperhomocysteinaemia, insulin resistance,metabolic syndrome, type-2 diabetes, menopause, aging, obesity, mentalstress, infections, inflammatory states, including periodontal disease,reumathoid arthritis, allograft vasculopathy and nitrate tolerance.

In yet another aspect of the method of the invention said condition ordisease is dyslipidemia, in particular hypercholesterolemia orhypertriglyceridemia, including dyslipidemia associated with low plasmalevel of HDL.

In yet another aspect of the method of the invention said condition ordisease to be treated or prevented is selected from the group consistingof: chronic cardiac failure, pulmonary hypertension, microvasculardiabetic complications, diabetic neuropathy, nephrotic syndrome, chronicrenal failure, adults respiratory distress syndrome (ARDS), cysticfibrosis, chronic obstructive pulmonary disease (COPD),preeclampsia/eclampsia, erectile dysfunction, Stein-Leventhal syndrome,sleep apnea, systemic lupus erythematosus, sickle cell anemia,non-specific inflammatory bowel diseases, gastric or duodenal ulcers,glaucoma, chronic liver disease, primary amyloidosis, andneurodegenerative diseases.

Said neurodegenerative disease can be in particular selected fromvascular dementia, Alzheimer's disease and Parkinson's disease.

In another variant said condition or disease can be in particularselected from gastric or duodenal ulcer wherein said compounds exertgastroprotective action.

In yet another aspect of the method of the invention said condition ordisease is liver injury wherein said compounds exert hepatoprotectiveaction.

Said liver injury can be in particular acute liver failure, chronicliver failure or liver cirrhosis.

Said liver injury can be also drug-induced liver injury caused by usingmedicaments, such as steroids, antifungal agents, antibiotics,immunosupressants, etc. The composition comprising said pyridiniumcompounds can be used prophylactically to protect liver in the case oftaking medicaments having hepatotoxic side effects. Said liver injurycan be also alcohol-induced liver injury caused by alcohol abuse, toxicliver injury caused by toxic chemicals, such as for example solvents,including carbon tetrachloride, chloroform, or by pesticides, or liverinjury caused by viral hepatitis.

The dosage will depend on the condition or disease being treated, thetype of treatment (therapeutic or preventive), the state and the age ofa patient being treated, and shall be finally determined individually bya medical practitioner. Generally, the amount of the pyridinium compoundor mixture of pyridinium compounds administered shall be in the range ofabout 0.1 to 10000 mg for administration in one single dose or individed doses, for example 0.5 mg to 1,125 mg, 1 mg to 1100 mg, 1.25 mgto 1075 mg, 1.5 mg to 1050 mg, 2.0 mg to 1025 mg, 2.5 mg to 1000 mg, 3.0mg to 975 mg, 3.5 mg to 950 mg, 4.0 mg to 925 mg, 4.5 mg to 900 mg, 5 mgto 875 mg, 10 mg to 850 mg, 20 mg to 825 mg, 30 mg to 800 mg, 40 mg to775 mg, 50 mg to 750 mg, 100 mg to 725 mg, 200 mg to 700 mg, 300 mg to675 mg, 400 mg to 650 mg, 500 mg, or 525 mg to 625 mg. In anotherembodiment the amount of the pyridinium compound(s) included in theadministered composition shall be from 0.1 mg to 25 mg. In someembodiments the amount of the pyridinium compound(s) included in theadministered composition shall be lower than 100 mg, or lower than 80mg, or lower than 60 mg, or lower than 50 mg, or lower than 30 mg, orlower than 20 mg, or lower than 10 mg, or lower than 5 mg, or lower than2 mg, or lower than 0.5 mg.

In the method of the invention pyridinium salts as defined above can beadministered as pharmaceutical compositions or diet supplements in aform for oral administration or ingestion. Such forms can includeconventional pharmaceutical. Forms for oral administration, such astablets, hard and soft capsules, powders, granules, solutions,suspensions, etc. They will include excipients (non-active ingredients)typically used in the art of formulation of pharmaceutical dosage forms,such as carriers, fillers, tabletting aids, flavour agents, etc. Each ofsuch excipients must be “acceptable”, i.e. must be compatible with otheringredients of the formulation, in particular with the active ingredientand must not be noxious for a patient. Non-limiting examples of suchmaterials which can be used as pharmaceutically acceptable carriers andfillers are: sugars, such as lactose, glucose and saccharose; starches,such as corn starch and potato starch; cellulose and cellulosederivatives, such as carboxymethylcellulose, ethylcellulose,hydroxypropylcellulose, cellulose acetate, microcrystalline cellulose;tragacanth; polyvinylpyrrolidone; calcium phosphate. Formulation fororal administration can also include lubricants and flow improvingagents, such as stearates, for example magnesium stearate, talc orsilica; disintegrating agents, such as for example sodium starchglycolate. Tablets can be coated with conventional coatings or sustainedrelease coatings.

Liquid formulations for oral administration include solutions, syrups orsuspensions. The suitable vehicle is water. They can include alsoconventional conservants, typically used for inhibition of microorganismgrowth, such as for example parabens, ascorbic acid, thimerosal, sorbicacid, methyl or propyl p-hydroxybenzoate, etc.

The compounds can be also administered parenterally in a form ofintravenous or subcutaneous injections. Suitable vehicles arepyrogen-free water, isotonic saline, phosphate buffers, Ringer'ssolution, oleaginous carriers, and other non-toxic substances used forthe formulation of pharmaceutical dosage forms. They can includeisotonicity agents, such as sodium chloride, sugars or polyalcohols,such as mannitol or sorbitol, and stabilizers or conservants.

The compounds can be also administered by inhalation, especially in thecase of the treatment of chronic obstructive pulmonary disease. Inparticular, the compounds can be administered by inhalation in a form ofa micronized powder or sprayed aerosol. They can be also administeredintranasally in a form of sprayed aerosol. Rectal administration of thecompounds in the method of the invention is also contemplated, in a formof creams, ointments or suppositories. In any case, the formulation willincorporate recipients, carriers and vehicles suitable for a givendosage form.

Dosage forms indicated above do not limit in any way the manner ofadministration of the pyridinium compounds or the compositionscontaining thereof.

The descriptions of typical and conventional formulations, techniques oftheir preparation and excipients can be for example found in theRemington: The Science and Practice of Pharmacy, 21 Ed., 2005.

For use in the method of the invention pyridinium compounds can beformulated as diet supplements. Pyridinium compounds can be inaccordance with the invention administered being added to nutritionalproducts of any form, typically in the amount of at least 5% by weightof the nutritional product, without being limited to the given contents.

As non-limiting examples, nutritional products can be in a form of aliquid, emulsion or paste, such as beverages, including soft drinks andalcoholic drinks, milk drinks, milk products such as yoghurts. Anotherexample can be solid products such as for example candies, chewing gums,jellies, chocolates, and powdered products, for example powdered drinksfor dissolution in water before consumption.

For use in the method of the invention pyridinium compounds can be alsoadded to vitamin supplements in a solid form, such as tablets, capsules,powder for dissolution, or a drink, containing besides the compound offormulae (I), (II) or (III) typical ingredients of vitamin supplements.

The examples below illustrate the use and pharmacological activity ofpyridinium compounds according to the invention.

EXAMPLE 1

The preparation of trigonelline thermolysate

Trigonelline thermolysate was prepared by thermal degradation(pyrolysis) of trigonelline at 220° C. for 30 minutes. Trigonelline (ina form of a thin layer of crystals) was pyrolysed in a thin-wall glassampoule filled with argon.

The resulting mixture of the pyrolysis products was dissolved in water,the solution was filtered and extracted with chloroform. Water wasdistilled off using rotary evaporator and the residue was dried overphosphorus pentoxide.

Preliminary analysis of the pyrolysis products (thermolysate) by meansof the mass spectrometry technique ToF-SIMS (Time of Flight SecondaryIon Mass Spectrometry) is presented below.

TABLE 1 SIMS analysis - composition of trigonelline thermolysate;identified compounds Product Structure m/e Peak intensity C₅H₅N

79 9265 C₅H₅NH

80 3305 C₅H₅NCH₃

94 480657 CH₃(C₅H₄NCH₃

108 14580 CH₃CH₃(C₅H₃)NCH₃

122 3472 COOH(C₅H₄NCH₃

138 87964 CH₃COOH(C₅H₃)NCH₃

152 25725 CH₃Npyr-pyrN

171 31922 CH₃Npyr-pyrNCH₃

186 2206 OHpyr-COOH(C₅H₃)NCH₃

231 30048

The major product of the pyrolysis is probably 1-methylpyridiniumcation, which is predominant in the spectrum. Other products present insignificant amounts are 1,2- and 1,4-dimethylpyridinium cations, andcations of: methyltrigonelline, (2′-hydroxypyridine)trigonelline,N-methyl(pyridine)-pyridinium and N-methyl(N′-methylpyridine)pyridinium.

EXAMPLE 2 Binding of Pyridinium Cations to Immobilized Heparin as anIndicator of Thrombolytic Responce

Research conducted by the inventors indicated a strong correlation ofthe binding of compounds of interest to heparin which could be relatedto the presence of the positively charged pyridinium cation in theirstructure, and the thrombolytic activity of the compounds. Neitherbinding nor thrombolytic activity were observed for uncharged pyridinederivatives, like for example other nicotinamide metabolites.

The presence of the heparin in the pyridinium salt solution changes theabsorption spectrum of the salt through the non-specific electrostaticinteractions of pyridinium cations with negatively charged heparinmoieties. The use of water-insoluble sepharose-immobilized heparin whichbinds some fraction of pyridinium cations causes the decrease of theconcentration of the salt in the solution. It should be emphasized thatsepharose immobilizing agent does not bind with pyridinium cations.

At the high excess of the immobilized heparin in relation to thepyridinium salt concentration the degree of binding depends only on theheparin concentration. It allows, by using the experimental methodologydescribed below, to estimate the degree of binding of differentpyridinium compounds to heparin, provided that analogical concentrationsof pyridinium salts and constant concentration of sepharose-immobilizedheparin are used.

Methodology of the Measurement of the Binding of Pyridinium Cations toImmobilized Heparin

To estimate a degree of binding of the pyridinium_compounds with heparinspectrophotometric method based on the absorption measurements ofaqueous solutions of tested compounds at 20÷100 μM before and after acontact with heparin was used.

Aqueous solutions of tested compounds were added to the suspension ofimmobilized heparin (Heparin Sepharose CL-6B, Amersham Biosciences AB,Sweden) (25 mg/mL) in ultrapure water from Millipore-Milli-Q system andincubated for 5 minutes. The suspensions of immobilized heparin with thetested compounds were then placed into Eppendorf microtubes andcentrifuged at 13 000 rpm for 4 minutes using FORCE 1418micro-centrifuge from Labnet Int. (Edison, N.J., USA). The UV-Visabsorption spectra of the resulting clear supernatants was measured withPerkin Elmer Lambda 40 spectrophotometer. All measurements wereperformed at room temperature.The degree of binding was estimated basedon comparison of integrated area under the absorption curve for thesolutions incubated and non-contacted with heparin.

Results for selected pyridinium compounds and nicotinamide metabolitesare presented in a Tables 2 and 3 below.

TABLE 2 The degree of binding of 3-substituted pyridinium salts andselected nicotinamide metabolites with known anti-thrombotic activity tosepharose immobilized heparin Degree of Thrombolytic Compound bindingresponse

1-methylnicotinamideMNA⁺ >45% yes

1-methyl-N′-(hydroxy-methyl)nicotinamideMNAF⁺ >35% yes

1-methyl-3-acetyl-pyridineMAP⁺ >25% yes

nicotinic acidNC >40% no

nicotinamide N-oxideN-OX >10% —

1-methyl-2-pyridone-5-carboxamide2-PYR  >5% no

nicotinamideNA  ≈0% no

TABLE 3 The degree of binding of trigonelline and its thermaldecomposition products to sepharose immobilized heparin Degree ofThrombolytic Pyridinium cation binding response

trigonellineTryg  ≈0% no

1-methylpyridineMP⁺ >25% yes

1,4-dimethylpyridine14-DMP⁺ >20% yes

1,2-dimethylpyridine12-DMP⁺ >15% —

4-methyltrigonelline4MeTryg  >5% —

The results presented in Table 2 show that quaternary pyridinium saltsdefined above have a tendency to bind to heparin, what may imply theircapability to interact with vascular endothelium surface and account fortheir therapeutic effect. The therapeutic effect, especially thethrombolytic response of the compounds presented in Table 2 is welldocumented (see patent WO 2005/067927 A2) and opposed to non-bindingproperties of other uncharged nicotinamide metabolites, which do notshow any significant anti-thrombotic activity.

Results presented in Table 3 indicate that quaternary pyridinium saltsshow a tendency to bind with heparin, what may imply their capability tointeract with vascular endothelium surface and account for theirtherapeutic effect.

It can be concluded that it is very likely to expect a strongthrombolytic response for all cationic products present in thetrigonelline thermolysate. In the case of two compounds,1-methylpyridine and 1,4-dimethyl-pyridine, their binding to heparin isaccompanied by anti-thrombotic effect in vivo. Although the simplecorrelation between these effects cannot be expected, the resultsobtained so far indicate the lack of thrombolytic response for compoundswhich do not bind to heparin. Essential factor in the non-specificelectrostatic interactions of polyanion with the cation is the presenceof the positive charge on nitrogen atom of the pyridinium ring.Compounds with sterically large substituents on ring nitrogen, forexample, propyl or benzyl groups, bind weaker with heparin in comparisonwith their N-methyl substituted analogs or do not bind at all. In thecase of zwitter-ionic compounds (e.g. trigonelline) the negative chargeof the molecule can neutralize the binding effect of positively chargepyridinium ring.

EXAMPLE 3

The measurement of thrombolytic effects of trigonelline thermolysate

Thrombolytic activity was assayed by the method developed originally byGryglewski et al., (Gryglewski R J, Korbut R, Ocetkiewicz A, Stachura J.In vivo method for quantitation for anti-platelet potency of drugs.Naunyn Schmiedebergs Arch Pharmacol 1978;302:25-30; Gryglewski R J 1978,Gryglewski et al., 2001). The scheme of the method is shown on FIG. 1.

Wistar rats (body weight of 300-350 g) were anaesthetised (thiopental 95mg/kg i.p) and heparinised (800 U/kg i.v.). Caniules were placed inarteries and connected from the left carotid artery vein with a pressuresensor and from the right carotid artery through heated (37° C.) line toperistaltic pomp. The blood from carotid artery was pumped to theextracorporeal circulation were it superfused (1.5 ml/min) a 3 cmcollagen strip excised from Achilles tendon hung on a spring-balancedauxotonic lever of a Harvard 386 isotonic transducer and the weight ofthe collagen strip was continuously registered. After superfusion theblood was returned to the circulation of an animal. During superfusion athrombus made of platelets aggregates stuck in the fibrin network(microscopically visualized by the Weigert method) was formed on thesurface of the collagen strip. A pressure and the thrombus weight werecontinuously recorded.

After 20-30 min of superfusion the weight of platelet-rich thrombusreached the plateau at the level of approximately 70-100 mg, and thislevel was steady for 3-5 hours as long as the blood superfusion lasted,unless the active compound was injected intravenously. Then thetrigonelline thermolysate, 1-methylpyridine or 1,4-dimethylpyridine wereinjected intravenously into the femoral vein (30 mg·kg⁻¹). Drug-inducedthrombolytic effect was observed as a decrease of the weight of thethrombus.

Intravenous injection of trigonelline thermolysate, prepared asdescribed in the example 1, induced an thrombolytic response in Wistarrats with extracorporeal circulation. Following a single injection oftrigonelline thermolysate (30 mg/kg) thrombolytic response was seen,reaching the plateau of 35±3.5% after 30 min and lasting atapproximately that level for 2-3 hours. 1-methylpyridine (30 mg/kg),being a major component of trigonelline thermolysate, also induced amoderate thrombolytic response. 1,4-dimethylpyridine induced athrombolytic response that was similar in magnitude and pattern to thatinduced by trigonelline thermolysate. In contrast to trigonellinethermolysate, 1-methylpyridine and 1,4-dimethylpyridine, trigonellineitself (30 mg/kg) did not induce significant thrombolytic response.Thrombolytic response of nicotinamide or nicotinic acid were of shortduration (shorter than 15-20 min) and their maxima were only 9±0.6% and5±0.9%, respectively. No thrombolytic response was seen fortrigonelline. Results of these experiments are shown on FIG. 2 to 5.

EXAMPLE 4

Antidiabetic Effects of Trigonelline Thermolysate in the Rat Model ofMetabolic Syndrome

Zucker fatty rats (fa/fa) represent widely used model of geneticallydetermined obesity, metabolic syndrome and dyslipidemia that mimicsmetabolic syndrome (also called insulin resistance syndrome) leading tothe development of manifest type-2 diabetes. The diagnostic symptom ofinsulin resistance is the elevated postprandial hyperglicemia that couldbe experimentally induced by glucose injection. In humans elevatedpostprandial hyperglicemia preceeds the development of type 2 diabetesand represent a prognostic factor for cardiovascular complications ofdiabetes. Elevated postprandial hyperglicemia can be observed also inZucker fatty rats (fa/fa).

Zucker fatty rats (fa/fa) (Crl:ZUC(Orl)—Lepra) at the age of 10 weekswere treated for 7 days with trigonelline thermolysate (100 mg/kg)(obtained as described in the example 1), with metformin (500 mg/kg) orwere left untreated (n=3-4 rats in each experimental group). After 7days glucose was injected at 2 g/kg (i.p.) and postprandialhyperglycemia was analyzed in the following periods: 0, 15, 30, 60 and90 minutes after the injection of glucose. As shown in FIG. 6, treatmentwith trigonelline thermolysate (100 mg/kg) decreased postprandialhyperglycemia induced by glucose injection to the similar degree as itwas the case with metformin (500 mg/kg). Accordingly, trigonellinethermolysate possesses antidiabetic properties.

Treatment with trigonelline thermolysate had no effect on the plasmalevels of triglycerides or free fatty acid, which were 3.5±0.2, 1.3±0.09mmol/l and 4.1±0.3, 1.4±0.07 mmol/l for control rats and rats treatedwith trigonelline thermolysate, respectively.

EXAMPLE 5

Hepatoprotective Effects of 1,4-dimethylpyridinium Chloride in MiceModel of Acute Liver Injury.

Balb/c mice at the age of 2 months were treated for 7 days with1,4-dimethylpyridinium chloride (100 mg/kg, per os) or were leftuntreated and then mice were injected with Concanavalin A (ConA)inducing T cell-mediated hepatitis. ConA was injected intravenously at adose of 20 mg/kg and 8 hours later liver injury was assessed on thebasis of transaminase (AST) level in plasma. As shown in FIG. 7,treatment with 1,4-dimethylpyridinium chloride (100 mg/kg) prevented arise in AST levels after Con A injection. Accordingly,1,4-dimethylpyridinium possesses hepatoprotective activity.

1. A method for the treatment or prevention of diseases or conditionsassociated with vascular endothelium dysfunction or liver injurycomprising the administration to a patient in a need of such treatmentor prevention of a therapeutically or prophylactically effective amountof a compound selected from the group consisting of:

wherein R represents hydrogen atom, CH₃, OH, pyridyl (C₅H₄N),1-methylpyridyl(C₅H₄N—CH₃) or pyridyl substituted with hydroxy group((OH)C₅H₃N), and X represents a physiologically acceptable counterion.2. The method according to claim 1 wherein said endothelium dysfunctionis accompanied by hypercholesteremia, hypertriglycerydemia or low HDLcholesterol level.
 3. The method according to claim 1 or 2 wherein saiddisease or condition is thrombosis or atherosclerosis.
 4. The methodaccording to claim 1 wherein said disease or condition is an acutecardiovascular event associated with atherosclerosis, in particularsudden cardiac death, acute coronary syndrome (including unstablecoronary artery disease, myocardial infarct), a condition whichnecessitates the coronary angioplasty (PCI) or coronary-aortal by-passsurgery (CABG), ischemic stroke, surgical treatment with extracorporealcirculation or a condition which necessitates a peripheral circulationrevascularization.
 5. The method according to claim 1 wherein saidcondition or disease is atherosclerosis in a patient with stablecoronary disease, brain ischemic episodes or atherosclerosis ofextremities, including Burger disease.
 6. The method according to claim1 wherein said condition or disease is selected from the risk factors ofatherosclerosis selected from the group consisting of:hypercholesterolemia, arterial hypertension, smoking,hyperhomocysteinaemia, insulin resistance, metabolic syndrome, diabetes,menopause, aging, obesity, mental stress, infections, inflammatorystates, including periodontal disease, reumathoid arthritis, allograftvasculopathy and nitrate tolerance.
 7. The method according to claim 1wherein said condition or disease is dyslipidemia, in particularhypercholesterolemia or hypertriglyceridemia, including dyslipidemiaassociated with low plasma level of HDL.
 8. The method according toclaim 1 wherein said condition or disease is thrombosis that is notrelated directly with atherosclerosis, in particular thrombosisassociated with implantation of metallic vascular prostheses (stents),coronary-aortal by-pass surgery(CABG), any type of surgery withextracorporeal circulation, hemodialysis, or venous thromboembolicdisease.
 9. The method according to claim 1 wherein said condition ordisease is selected from the group consisting of: chronic cardiacfailure, pulmonary hypertension, microvascular diabetic complications,diabetic neuropathy, nephrotic syndrome, chronic renal failure, adultsrespiratory distress syndrome (ARDS), cystic fibrosis, chronicobstructive pulmonary disease (COPD), preeclampsia/eclampsia, erectiledysfunction, Stein-Leventhal syndrome, sleep apnea, systemic lupuserythematosus, sickle cell anemia, non-specific inflammatory boweldiseases, gastric or duodenal ulcers, glaucoma, chronic liver disease,primary amyloidosis, and neurodegenerative diseases.
 10. The methodaccording to claim 9 wherein said neurodegenerative disease is selectedfrom vascular dementia, Alzheimer's disease and Parkinson's disease. 11.The method according to claim 9 wherein said condition or disease isgastric or duodenal ulcer.
 12. The method according to claim 1 whereinsaid condition or disease is liver injury.
 13. The method according toclaim 12 wherein said liver injury is acute liver failure, chronic liverfailure or liver cirrhosis.
 14. The method according to claim 13 whereinsaid liver injury is drug-induced liver injury, alcohol-induced liverinjury, toxic liver injury, or liver injury caused by viral hepatitis.15. The method according to claim 1 wherein said administration is viaoral route.
 16. The method according to claim 1 wherein saidadministration is via parenteral route.
 17. The method according toclaim 1 wherein said compound has the formula (I)

wherein R represents hydrogen atom or CH₃ in the position 2 or 4 of thepyridine ring.
 18. The method according to claim 17 wherein R representsCH₃ in the position 4 of the pyridine ring.
 19. A method for thetreatment or prevention of diseases or conditions associated withvascular endothelium dysfunction or liver injury comprising theadministration to a patient in a need of such treatment or prevention ofa therapeutically or prophylactically effective amount of a1,4-dimethylpyridine salt of the formula

wherein X represents a physiologically acceptable counterion.
 20. Themethod of claim 19 wherein X represents chloro.
 21. A method for thetreatment or prevention of atherosclerosis and/or thrombosis comprisingthe administration to a patient in a need of such treatment orprevention of a therapeutically or prophylactically effective amount ofa 1,4-dimethylpyridine salt of the formula

wherein X represents a physiologically acceptable counterion.
 22. Themethod of claim 21 wherein X represents chloro.
 23. A method for thetreatment or prevention of a liver injury comprising the administrationto a patient in a need of such treatment or prevention of atherapeutically or prophylactically effective amount of a1,4-dimethylpyridine salt of the formula

wherein X represents a physiologically acceptable counterion.
 24. Themethod of claim 23 wherein X represents chloro.